GDNF and ribozymes for retinitis pigmentosa

常染色体显性遗传性视网膜色素变性(ADRP)是一组显性遗传性视网膜变性疾病。它是慢性神经退行性疾病的典型，因为ADRP的特征是视杆感光细胞的渐进性凋亡丢失，通常导致缓慢但不可阻挡的失明进展。在之前的资助期间，用腺相关病毒(AAV)递送的核酶在一个啮齿动物ADRP模型中阻止了这种退化。结果是接受治疗的视杆细胞长期存活并保留了视觉功能 眼睛。在这个项目中，我们建议改进和扩展这些结果，以推动视网膜基因治疗走向临床应用。对于缓慢进展的五种神经退行性疾病，重要的是评估任何治疗效果的持久性，确定是否可以进一步改善疗效以增强安全性，并将该治疗方式应用于具有人类眼睛大小的大型动物疾病模型。为达致这些目标，我们建议：(1)量度 该研究旨在研究AAV介导的核酶对P23H视紫红质大鼠的视网膜神经保护作用的持续时间，并在这些大鼠一岁时开始治疗，此时只剩下5-15%的正常视杆功能，以便在最初的人体试验中模拟预期情况，晚期RP患者将成为第一个接受测试的患者。(2)通过(A)提高核酶的催化活性、稳定性和表达水平，(B)通过增强 从AAV载体中表达胶质细胞源性神经营养因子(GDNF)，以及(C)确定相对于单基因治疗而言，通过表达GDNF和核酶是否提高了光感受器的存活。要测试的元素包括核酶结构改进、RNA稳定元件、替代启动子、替代AAV血清型和针对视网膜和视网膜相关细胞类型的新亚群的载体衣壳突变体。 不重叠的改进将被组合成单一的载体(或载体的组合)，以生产最佳的治疗试剂。(3)检测AAV载体核酶对ADRP和X连锁RP犬模型的抗突变作用。这包括T4R视蛋白突变(视蛋白第4位的精氨酸取代苏氨酸)，导致与人类相似的显性RP，以及犬RPGR基因突变，导致狗与人类类似的X-连锁视网膜疾病。我们在这里的主要目标是确定 在小啮齿类动物的眼睛中成功地进行了治疗，有效地将其扩展到了人类大小的眼睛。如果成功，我们预计这些优化的、更通用的核酶方法将允许我们尝试对拥有导致RP的广泛突变的人类进行基因治疗。

Autosomal Dominant Retinitis Pigmentosa (ADRP) comprises a group of dominantly inherited degenerative retinal diseases. It is prototypic of slow neurodegenerative diseases in that ADRP is characterized by the progressive apoptotic loss of rod photoreceptor cells normally leading to a slow but inexorable progression to blindness. In the previous grant period ribozymes delivered by Adeno-associated virus (AAV) were employed to block this degeneration in one rodent ADRP model. The result was long-term rod cell survival and preservation of visual function in treated eyes. In this project we propose to improve and extend those results in order to propel retinal gene therapy towards a clinical application. For slowly progressive neurodegenerafive diseases it is important to assess the longevity of any therapeutic effect, to determine if efficacy can be further improved to enhance safety and to apply the therapeutic modality to a large animal disease model with human sized eye. To achieve these ends, we propose to: (1) Measure the duration of neural retina protection afforded by AAV-delivered ribozymes in P23H rhodopsin rats, and to initiate therapy in these rats at one year of age when only 5-15% of normal rod function remains in order to model the expected situation in an initial human trial where late-stage RP patients will be the first tested. (2) Improve in vivo ribozyme performance by (a) by increasing ribozyme catalytic activity, stability and expression level, (b) by enhancing the expression of glial cell derived neurotrophic factor (GDNF) from AAV vectors and (c) by determining whether photoreceptor survival is enhanced by expressing GDNF along with the ribozyme relative to single gene therapy. Elements to be tested include ribozyme structural improvements, RNA stability elements, alternative promoters, alternative AAV serotypes and vector capsid mutants that target novel subsets of retinal and retina-associated cell types. Non-overlapping improvements will be combined into a single vector (or combination of vectors) to produce the optimal therapeutic reagent. (3) Test AAV-vectored ribozymes against mutations creating canine models of ADRP and X-linked RP. This includes the T4R rod opsin mutation (substitution of arginine for threonine at position 4 of rod opsin) leading to dominant RP similar to humans with the T4K mutation and mutations in the canine RPGR gene that cause X-linked retinal disease in dogs analogous to that in humans. Our primary aim here will be to determine whether successful therapy in the small rodent eye scales effectively to a human sized eye. If successful, we anticipate that these optimized, more general ribozyme approaches will permit us to attempt gene therapy in humans possessing a wide spectrum of mutations causing RP.